To understand the interactions between the Mycobacterium tuberculosis (mtb) antigen 85 enzymes and their substrates, identify components of each enzyme that lead to differences in substrate specificity, identify inhibitors of the catalytic activity, and combine these data to develop novel lead compounds for drug development. The antigen 85 (Ag85) enzymes catalyze the final step required for synthesis of the highly hydrophobic cell envelope of mycobacteria. In vivo studies suggest the three Ag85 enzymes of mtb are responsible for synthesizing different cell envelope components, trehalose dimycolate (TDM) or mycolyl-arabinogalactan (mAG). However, it is not clear how the three Ag85 homologs achieve this substrate specificity and selectivity. In this proposal, we combine biochemical, biophysical, and structural approaches to address this question. This research project is separated into 4 separate but interrelated aspects. First, we will use steady-state kinetics and isothermal titration calorimetry to quantify the enzymatic reaction and binding interactions between the antigen 85 enzymes and their substrates. This will further refine our understanding the different roles these three enzymes play in the bacterium. The second set of experiments uses X-ray crystallography to directly visualize structural changes that occur in the enzymes during catalysis. By obtaining these snapshots of each step in the reaction, we can obtain unprecedented detail about the antigen 85/substrate interactions. The third portion of this study examines the affect of mutations in the enzymes to probe the affects of structural changes on the enzymatic reaction and interactions with the different substrates used by these enzymes. Steady-state kinetics, binding studies, and X-ray crystallography will all be used to examine these mutants. The final method of inquiry will develop novel inhibitors of the antigen 85 enzymes. This will offer two benefits. First, these compounds will be used to study the thermodynamics of complex formation and allow comparison of the three antigen 85 enzymes. Second, these compound represent leads for the development of new therapeutics for the treatment of tuberculosis, lepresy, M. ulcerans infections, and the opportunistic infection by M. avium specied of people with suppressed immune systems. PUBLIC HEALTH RELEVANCE: Tuberculosis kills more people worldwide than any other treatable disease. Typical treatment with drugs lasts 6 months. Better drugs for treating tuberculosis are desperately needed. Understanding enzyme function in these pathogenic bacteria is central to developing new drugs that specifically inhibit those enzymes and kill the bacteria. This study attempts to better understand the function of 3 tuberculosis enzymes that are important for creating the protective outer layer of the bacteria that cause tuberculosis as a basis for designing new and better drugs to treat tuberculosis.